AUTHOR=Kaewmee Saowalak , Mano Chonlada , Phanitchakun Thanari , Ampol Rinnara , Yasanga Thippawan , Pattanawong Urassaya , Junkum Anuluck , Siriyasatien Padet , Bates Paul A. , Jariyapan Narissara 

TITLE=Natural infection with Leishmania (Mundinia) martiniquensis supports Culicoides peregrinus (Diptera: Ceratopogonidae) as a potential vector of leishmaniasis and characterization of a Crithidia sp. isolated from the midges

JOURNAL=Frontiers in Microbiology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2023.1235254

DOI=10.3389/fmicb.2023.1235254

ISSN=1664-302X

ABSTRACT=<p>The prevalence of autochthonous leishmaniasis in Thailand is increasing but the natural vectors that are responsible for transmission remain unknown. Experimental <italic>in vivo</italic> infections in <italic>Culicoides</italic> spp. <italic>with Leishmania</italic> (<italic>Mundinia</italic>) <italic>martiniquensis</italic> and <italic>Leishmania</italic> (<italic>Mundinia</italic>) <italic>orientalis</italic>, the major causative pathogens in Thailand, have demonstrated that biting midges can act as competent vectors. Therefore, the isolation and detection of <italic>Leishmania</italic> and other trypanosomatids were performed in biting midges collected at a field site in an endemic area of leishmaniasis in Tha Ruea and a mixed farm of chickens, goats, and cattle in Khuan Phang, Nakhon Si Thammarat province, southern Thailand. Results showed that <italic>Culicoides peregrinus</italic> was the abundant species (&gt;84%) found in both locations and only cow blood DNA was detected in engorged females. Microscopic examination revealed various forms of <italic>Leishmania</italic> promastigotes in the foregut of several <italic>C. peregrinus</italic> in the absence of bloodmeal remnants, indicating established infections. Molecular identification using ITS1 and 3’UTR <italic>HSP70</italic> type I markers showed that the <italic>Leishmania</italic> parasites found in the midges were <italic>L. martiniquensis</italic>. The infection rate of <italic>L. martiniquensis</italic> in the collected flies was 2% in Tha Ruea and 6% in Khuan Phang, but no <italic>L. orientalis</italic> DNA or parasites were found. Additionally, organisms from two different clades of <italic>Crithidia</italic>, both possibly new species, were identified using SSU rRNA and gGAPDH genes. Choanomastigotes and promastigotes of both <italic>Crithidia</italic> spp. were observed in the hindgut of the dissected <italic>C. peregrinus</italic>. Interestingly, midges infected with both <italic>L. martiniquensis</italic> and <italic>Crithidia</italic> were found. Moreover, four strains of <italic>Crithidia</italic> from one of the clades were successfully isolated into culture. These parasites could grow at 37°C in the culture and infect BALB/c mice macrophages but no multiplication was observed, suggesting they are thermotolerant monoxenous trypanosomatids similar to <italic>Cr. thermophila.</italic> These findings provide the first evidence of natural infection of <italic>L. martiniquensis</italic> in <italic>C. peregrinus</italic> supporting it as a potential vector of <italic>L. martiniquensis</italic>.</p>